Cholesterol Enrichment of Human Monocyte/Macrophages Induces

Cholesterol Enrichment of Human Monocyte/Macrophages
Induces Surface Exposure of Phosphatidylserine and the
Release of Biologically-Active Tissue
Factor–Positive Microvesicles
Ming-Lin Liu, Michael P. Reilly, Peter Casasanto, Steven E. McKenzie, Kevin Jon Williams
Downloaded from http://atvb.ahajournals.org/ by guest on October 1, 2016
Objective—Biologically significant amounts of two procoagulant molecules, phosphatidylserine (PS) and tissue factor
(TF), are transported by monocyte/macrophage-derived microvesicles (MVs). Because cellular cholesterol accumulation
is an important feature of atherosclerotic vascular disease, we now examined effects of cholesterol enrichment on MV
release from human monocytes and macrophages.
Methods and Results—Cholesterol enrichment of human THP-1 monocytes, alone or in combination with lipopolysaccharide (LPS), tripled their total MV generation, as quantified by flow cytometry based on particle size and PS exposure.
The subset of these MVs that were also TF-positive was likewise increased by cellular cholesterol enrichment, and these
TF-positive MVs exhibited a striking 10-fold increase in procoagulant activity. Moreover, cholesterol enrichment of
primary human monocyte-derived macrophages also increased their total as well as TF-positive MV release, and these
TF-positive MVs exhibited a similar 10-fold increase in procoagulant activity. To explore the mechanisms of enhanced
MV release, we found that cholesterol enrichment of monocytes caused PS exposure on the cell surface by as early as
2 hours and genomic DNA fragmentation in a minority of cells by 20 hours. Addition of a caspase inhibitor at the
beginning of these incubations blunted both cholesterol-induced apoptosis and MV release.
Conclusions—Cholesterol enrichment of human monocyte/macrophages induces the generation of highly biologically
active, PS-positive MVs, at least in part through induction of apoptosis. Cholesterol-induced monocyte/macrophage
MVs, both TF-positive and TF-negative, may be novel contributors to atherothrombosis. (Arterioscler Thromb Vasc
Biol. 2007;27:430-435.)
Key Words: macrophages 䡲 microparticles 䡲 tissue factor 䡲 apoptosis 䡲 atherosclerosis
M
icrovesicles (MV) are small membranous structures
that are released from cells on activation or during
apoptosis.1,2 Monocyte/macrophage-derived MVs transport
biologically significant amounts of phosphatidylserine (PS), a
membrane lipid that enhances clot propagation,3 and tissue
factor (TF), a potent initiator of coagulation in vivo.1,4,5 Based
on several observations, we hypothesized a link between
cellular cholesterol enrichment and monocyte/macrophage
MV generation. First, clinical studies have shown an association between hyperlipidemia and elevated numbers of circulating monocyte/macrophage-derived PS-positive MVs,6 –9
and monocyte/macrophage-derived MVs have been found in
human atherosclerotic lesions as well.4,10 Second, monocyte/
macrophages accumulate unesterified cholesterol (UC) in
vivo during atherogenesis,11 presumably owing to their up-
take of lipoproteins that have been retained and modified
within the arterial wall.12–14 Third, cholesterol loading of
primary human monocyte-derived macrophages (MDM) in
vitro was reported to stimulate the expression of TF,15 and
increased TF expression was found in atherosclerotic vessels
from cholesterol-fed rabbits,16,17 in human lipid-rich plaque
macrophages,18 and in monocytes from hypercholesterolemic
patients.19 Moreover, our group recently reported that hyperlipidemia induces a prothrombotic state in vivo in animals,20
although none of these aforementioned studies examined
TF-positive MV release. Fourth, plasma lipid lowering by
medication,17 diet,21,22 or extracorporeal low-density lipoprotein apheresis23 reduced TF levels in vivo, and extensive
cholesterol depletion of monocytes in vitro for the purpose of
disrupting normal plasma membrane structure was recently
Original received February 17, 2006; final version accepted November 20, 2006.
From the Dorrance H. Hamilton Research Laboratories (M.-L.L., K.J.W.), Division of Endocrinology, Diabetes and Metabolic Diseases, and the
Cardeza Foundation for Hematologic Research (M.P.R., P.C., S.E.M.), Division of Hematology, Department of Medicine, Jefferson Medical College of
Thomas Jefferson University, Philadelphia, Pa.
A preliminary report of this work was presented at 6th Annual Conference on Arteriosclerosis, Thrombosis and Vascular Biology, Washington, DC,
30 April 2005.
Correspondence to Kevin Jon Williams or Ming-Lin Liu, Division of Endocrinology, Thomas Jefferson University, 1020 Locust Street, Suite 348,
Philadelphia, PA 19107. E-mail K_Williams@mail.jci.tju.edu or Ming-lin.Liu@jefferson.edu
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
430
DOI: 10.1161/01.ATV.0000254674.47693.e8
Liu et al
shown to inhibit their calcium ionophore-stimulated release
of total MVs.24 Nonetheless, direct effects of cholesterol
enrichment on monocyte/macrophage MV release have not
been reported. In the current study, we directly loaded human
THP-1 monocytic cells and primary human MDMs with
exogenous cholesterol and assessed their vesiculation, as well
as the biologic activity of the released MVs. LPS, a wellknown stimulus for MV generation by THP-1 monocytes,25
served as a positive control.
Methods
Cell Culture and Cholesterol Treatment
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THP-1 cells (ATCC, Manassas, Va) were maintained in suspension
culture in RPMI-1640 supplemented with 7% FBS, 1% penicillin/
streptomycin, and 0.05 mmol 2-mercaptoethanol/L, as recommended
by the ATCC. Before each experiment, cells were transferred to
serum-free RPMI-1640 containing 0.2% BSA (RPMI/BSA), followed by a 1-hour preincubation at 37°C. Next, the cells were
incubated at 37°C without (control) or with 10 ␮g unesterified
cholesterol (UC)/mL, with or without 10 ␮g LPS/mL, for 0 to 20
hours in serum-free RPMI/BSA. The UC was delivered as a
water-soluble complex with methyl-␤-cyclodextrin (UC/mcd; 1:6
molar ratio of UC:mcd; Sigma), which has been widely used to
modify the cholesterol content of cultured cells without potentially
confounding effects from receptor engagement.26 –28 We verified that
all UC/mcd preparations contained no detectable endotoxin by the
Limulus amoebocyte lysate assay (Associates of Cape Cod, Inc).
Adherent primary human MDMs were prepared from fresh Buffy
coats by low-speed Ficoll density gradient centrifugation to purify
the mononuclear cell fraction, followed by positive selection of
CD14⫹ monocytes using magnetic beads coated with a monoclonal
anti-CD14 antibody (Miltenyi Biotec Inc.). The CD14⫹ monocytes
were differentiated into macrophages by a 7-day incubation at 37°C
in DMEM supplemented with 10% FBS, 10% horse serum, 0.5 ng
granulocyte-macrophage colony stimulating factor (CSF)/mL, and
0.5 ng macrophage CSF/mL.29,30 Because concentrations of UC/mcd
up to 380 ␮g UC/mL of medium, based on the mass of cholesterol
in the UC/mcd complexes, have been used to cholesterol-enrich
primary macrophages,26 we chose an intermediate concentration, 100
␮g UC/mL, for our studies. Adherent MDMs were incubated at 37°C
for 20 hours without (control) or with UC/mcd in RPMI/BSA.
Characterization of MVs and Cells
For flow cytometry, we left the THP-1 cells, which grow in
suspension, in their conditioned media. At each time point, samples
of cells and media were fixed by addition of paraformaldehyde
(PFA) to a final concentration of 2%, followed by storage at ⫺80°C
until analysis, as described.31,32 For adherent primary human MDMs,
supernatants without cells were fixed in 2% PFA, whereas the cell
monolayers were scraped and fixed in suspension in 1% PFA and
then 70% ethanol. Consistent with prior literature,31,32 our initial
studies indicated that PFA fixation of MVs and cells protects them
from changes induced by a single freezing and thawing. Moreover,
we specifically verified that the cholesterol-induced increase in MV
generation measured after fixation and freezing was the same as that
seen using unfixed, unfrozen material (data not shown). The number
of MVs in each sample was quantified by flow cytometry, using
gating criteria based on particle size, as detected by forward scatter,
and surface exposure of PS, as detected by staining with phycoerythrin (PE)-labeled annexin-V (BD Pharmingen).25 The portion of
these PS-positive MVs that were also TF-positive was quantified by
simultaneous staining with a fluorescein isothiocyanate (FITC)labeled anti-human TF monoclonal antibody (Cat# 4508CJ, American Diagnostica). For THP-1 cell suspensions, results are reported as
MVs per 1000 cells. Because MDMs are adherent, we added 25 000
15-␮m latex beads (Molecular Probes) to each 100 ␮L of their
conditioned medium as a reference, and our results are reported as
absolute numbers of MVs per ml of conditioned culture medium.
Cholesterol-Induced Microvesicle Release
431
Cellular cholesterol content was quantified in isopropanol extracts of
unfixed THP-1 cells by the cholesterol oxidase method (Wako
Inc).33 In some samples, late-stage apoptosis of THP-1 monocytes or
primary human MDMs was assessed by terminal deoxynucleotidyl
transferase FITC-dUTP nick end labeling of the cells (TUNEL;
APO-DIRECT kit, BD Pharmingen), followed by flow cytometric
quantification. For causal studies of the role of apoptosis, a subset of
incubations included a caspase-3 inhibitor, Z-DEVD-FMK, at the
manufacturer’s highest recommended concentration (100 ␮mol/L;
R&D Systems).
Assessments of Tissue Factor Antigen and Tissue
Factor Procoagulant Activity
Cellular release of TF antigen was quantified by ELISA. Culture
suspensions (THP-1 monocytes) or conditioned media (primary
human MDMs) were centrifuged at low speed (200g for 10 minutes)
to remove cells or large debris while leaving MVs in the supernatant.
Tissue factor concentrations were then determined (IMUBIND
Tissue Factor ELISA kit, American Diagnostica).
Tissue factor procoagulant activity was measured with fresh
(unfixed, unfrozen) samples using a chromogenic assay for the
activation of clotting factor X (active factor Xa) by MVs captured by
platelets, as previously described.24 In brief, MV-containing culture
supernatants were obtained by low-speed centrifugation of conditioned medium from THP-1 cells or MDMs after treatment without
or with UC. Washed human platelets were activated with 5 ␮g
collagen/mL and then incubated with these MV-containing supernatants for 1 hour at 37°C with continuous rocking. Unbound MVs
were then removed by sedimenting the platelets and captured MVs at
1600g for 10 minutes at RT and discarding the supernatants. The
pelleted platelets with bound MVs were then resuspended in buffer
containing CaCl2, exogenous factor VIIa, and inactive factor X
(American Diagnostica) and incubated at 37°C for 30 minutes. To
stop the reaction, platelets with bound MVs were removed by
filtration (0.2 ␮m). The filtrate, which contained any activated factor
Xa, was portioned into aliquots in a 96-well plate and mixed with a
chromogenic Xa substrate, Spectrozyme-Xa (0.5 mmol/L, American
Diagnostica). After 60 minutes at 37°C, absorbances at 405 nm were
recorded in a microplate reader. As negative controls, TF activity
was measured in samples in which factor VIIa, factor X, or
Spectrozyme-Xa had each been omitted from the reaction. Lipidated
TF (American Diagnostica) was used as standards (0, 10, 20, and 40
pmol/L). We also measured TF procoagulant activity of washed
MVs, which were isolated from 1 mL of THP-1 culture supernatant
by high-speed centrifugation (100 000g at 4°C for 1 hour), then
washed and resuspended in 1 mL RPMI/BSA for analysis of activity,
as above.
Statistical Analyses
Values are shown as means⫾SEM, n⫽4 to 5. Comparisons among
several groups were performed using one-way analysis of variance
(ANOVA), followed by the Student-Newman-Keuls test, with
P⬍0.05 considered significant. Comparisons between two groups
used Student unpaired t test.
Results and Discussion
We found that cholesterol enrichment of human THP-1
monocytes provoked a significant increase in the release of
PS-positive MVs (Figure 1A and 1B). The effect was evident
as early as 2 hours and persisted for the entire 20-hour period
we examined (Figure 1A and 1B). By 20 hours, the total MV
count from cholesterol-enriched THP-1 cells was nearly 3
times the value from untreated control cells. As a positive
control,25 LPS also stimulated THP-1 monocyte MV release,
but we now found that cholesterol given simultaneously with
LPS increased microvesiculation at each time point as compared with either agent alone (Figure 1B). To establish that
cholesterol was responsible for these effects, we verified that
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Arterioscler Thromb Vasc Biol.
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Figure 1. Cholesterol enrichment of monocytic cells increases
total MV generation. Suspensions of THP-1 monocytes were
treated for 0 to 20 hours at 37°C with UC, LPS, both, or neither
(Control), as indicated, and then samples were analyzed by flow
cytometry. A, Representative dot-plots of forward scatter (FSC,
indicating size) vs annexin V-PE staining (indicating PS exposure) for suspensions of control and UC-enriched cells. The
squares enclose the population of PS-positive MVs. The ovals
indicate cellular populations with the same size range and PE
intensity as control THP-1 cells at t⫽0 hours, whereas the circles identify a population of cells that appeared over time, particularly with cholesterol enrichment. B, Quantitative counts of
PS-positive MVs generated over time under the four experimental conditions. P⬍0.01 by ANOVA. *P⬍0.05, †P⬍0.01, and
‡P⬍0.001 vs the control group by the Student-Newman-Keuls
test.
incubation with UC/mcd caused significant cholesterol enrichment of THP-1 monocytes throughout the 20-hour timecourse (51.1⫾4.0 at 0 hours, 86.3⫾12.7 at 2 hours, and
123.0⫾13.2 ␮g cholesterol/mg cell protein at 20 hours, n⫽4,
P⬍0.01 for 2 hours and for 20 hours versus 0 hours). Similar
cholesterol enrichment was seen in cells incubated with the
combination of UC and LPS, whereas no changes in cellular
cholesterol content were observed in the control or LPS-only
groups (data not shown). Furthermore, media supplemented
with the same low concentration of methyl-␤-cyclodextrin
(0.15 mmol/L), but not complexed to cholesterol, failed to
measurably affect THP-1 MV generation over time (eg, the
MV count after 20 hours in the presence of methyl-␤cyclodextrin without UC was 93%⫾3% of the control value
in unsupplemented medium, NS).
In addition to increasing total cellular output of MVs,
cholesterol loading of THP-1 cells in the absence or
presence of LPS caused large increases in the cellular
release of TF-positive MVs, as measured either by flow
cytometry of the cell culture suspension (Figure 2A), or by
TF ELISA of cell-culture supernatants (supplemental Fig-
Figure 2. Cholesterol enrichment of monocytic cells increases
the generation of biologically active TF-positive MVs. Suspensions of THP-1 monocytes were treated as in Figure 1. A, Quantitative counts at 4 hours of PS-positive MVs that were also
TF-positive. B, TF procoagulant activity of MV-containing supernatants in an assay using exogenous factor VIIa and collagenactivated platelets. P⬍0.01 by ANOVA. *P⬍0.05, †P⬍0.01, and
‡P⬍0.001 vs the control group by the Student-Newman-Keuls test.
ure I, available online at http://atvb.ahajournals.org). Importantly, cellular cholesterol loading of THP-1 cells
caused a remarkable 10-fold increase in the TF procoagulant activity of MV-containing cell culture supernatants
(Figure 2B) and of washed THP-1 MVs (not shown),
consistent with the increase in TF mass (supplemental Figure I)
and possibly TF de-encryption (see references34 –36). To
examine these effects in a primary cell, we found that
cholesterol-enrichment of human MDMs caused large, statistically significant increases in total (Figure 3A) and TFpositive (supplemental Figure II) MV release compared with
control. Moreover, MV-containing cell culture supernatants
from cholesterol-enriched primary human MDMs also
showed a striking 10-fold increase in TF procoagulant activity over control (Figure 3B). These results suggest that
enhanced release of procoagulant MVs from cholesterolenriched monocyte/macrophages could contribute to the general prothrombotic state in hypercholesterolemia,19,20,23,37 and
to the prothrombotic interior of lipid-rich, vulnerable
plaques.4,5,10,14
The flow cytometry protocol for THP-1 monocytes allowed us to simultaneously examine MVs and the cells that
generated them. Our data showed no significant changes in
Liu et al
Cholesterol-Induced Microvesicle Release
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Figure 4. Apoptosis during cholesterol-induced MV generation.
THP-1 monocytes were incubated at 37°C with or without UC,
LPS, or combinations, as indicated, then analyzed by flow
cytometry. Displayed are representative histograms of TUNEL
staining after 0 hours (red), 2 hours (black), 4 hours (green), and
20 hours (blue). Each histogram includes approximately 1000
cells. Percentages indicate the proportion of cells that were
TUNEL positive at 20 hours.
Figure 3. Cholesterol enrichment of primary human monocytederived macrophages increases the generation of total as well
as biologically-active TF-positive MV. Adherent primary human
MDMs in 6-well plates were treated without (Control) or with UC
for 20 hours. A, Total concentrations of PS-positive MVs in the
conditioned medium, as assessed by flow cytometry. B, TF procoagulant activity of MV-containing culture supernatants.
‡P⬍0.001 vs the control group by Student unpaired t test.
either cell-surface exposure of PS or cellular size when
THP-1 cells were incubated in unsupplemented serum-free
medium (Figure 1A and supplemental Figure III) or treated
with LPS (supplemental Figure III; size data not shown).
Under these conditions, a low level of cellular PS exposure
was seen in the current (Figure 1A) and previous studies25
which could play a role in generation of PS-positive MVs by
these cells.38,39 In contrast, cholesterol enrichment caused a
majority of the THP-1 monocytes to promptly display strong
staining for PS on their surface by 2 hours (Figure 1A and
supplemental Figure III), and to gradually shrink over time,
as shown by a downward shift in forward scatter (Figure 1A).
Because cell-surface exposure of PS not only induces procoagulant responses5 but may also indicate early-stage apoptosis,5,40 we examined a definitive marker of late-stage apoptosis, namely DNA fragmentation by TUNEL. At the 20-hour
time point, the proportions of TUNEL-positive cells were
only 1% to 2% in serum-free medium, and only 2% to 3%
with LPS stimulation, but rose to approximately 30% with
cholesterol enrichment of THP-1 cells (Figure 4). Likewise,
the proportions of primary human MDMs that were TUNELpositive were only 1% in serum-free medium but rose to
approximately 11% after 20 hours of cholesterol enrichment.
Thus, similar to prior literature,26 we saw significant cholesterol-induced apoptosis, which raises the possibility that at
least some of the cholesterol-induced MV generation in our
system might be from apoptotic blebbing. Interestingly, our
human monocyte/macrophages underwent UC-induced apoptosis even in the absence of other manipulations (see
reference26), indicating an unexpectedly robust response in
these cells.
To assess a causal role for apoptosis, we used the caspase-3
inhibitor Z-DEVD-FMK. Addition of this inhibitor during
cholesterol enrichment of THP-1 monocytes delayed cellsurface exposure of PS by 2 to 4 hours, but did not affect the
final percentage of PS-positive cells at 20 hours (Supplemental Figure IVA). The inhibitor significantly decreased the
percentage of TUNEL-positive cells at 20 hours from
28⫾2.8% to 12.5⫾2.2% (supplemental Figure IVB,
P⬍0.001). Importantly, the caspase-3 inhibitor significantly
decreased the number of PS-positive MVs released during
UC-enrichment over time, although not entirely to the level of
unenriched control cells at 6 hours and 20 hours (Figure 5 and
supplemental Figure IVA). In contrast, we did not see any
effect of the caspase-3 inhibitor on the number of TF-positive
MVs released after UC-enrichment (data not shown), suggesting that TF synthesis and TF-positive MV release may
come from a subset of these cells that are not in late-stage
apoptosis (see reference41).
Taken together, our results directly link cellular cholesterol
enrichment, a known consequence of hyperlipidemia, arterial
retention of lipoproteins, and atherogenesis,12,14 with the
enhanced generation of biologically active monocyte/macrophage-derived MVs, at least in part through induction of
apoptosis. Related effects may occur with other cell types,
though independent of apoptosis.42 The effects of TF-positive
monocyte/macrophage-derived MVs and their potential con-
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Arterioscler Thromb Vasc Biol.
February 2007
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Figure 5. Role for apoptosis in cholesterol-induced MV generation. THP-1 monocytic cells were incubated at 37°C without or
with UC, Z-DEVD-FMK (a caspase-3 inhibitor), or combinations,
as indicated, then analyzed by flow cytometry. Displayed are
quantitative counts of PS-positive MVs over time after the indicated treatments. P⬍0.01 by ANOVA. Symbols labeled with different lowercase letters are statistically different by the StudentNewman-Keuls test (P⬍0.05).
tributions to atherothrombosis have been investigated in the
present as well as in previous studies.4,43 Nevertheless, more
than 80% of the MVs in our study (Figures 1B and 2A; Figure
3A and supplemental Figure II) and in prior literature44 were
PS-positive but TF-negative. We speculate that these particles
may be biologically active as well, for example, by enhancing
clot propagation,3,5 causing apoptosis of nearby cells,45,46
blocking PS receptors that could otherwise promote phagocytosis of apoptotic bodies,47 and altering gene expression via
their display of PS48 and perhaps other biologically active
molecules.49,50 Of interest, we recently found that injection of
cholesterol-induced monocyte MVs into rats provoked substantial leukocyte rolling and adherence to post-capillary
venules in vivo, indicative of endothelial activation (Liu M-L,
Scalia R, Williams KJ, unpublished data, 2006). Thus,
cholesterol-induced PS-positive monocyte/macrophage MVs,
both TF-positive and TF-negative, may be novel contributors
to atherothrombosis. Because cholesterol is an exchangeable
molecule that can be removed by acceptor particles, such as
high-density lipoprotein, cellular production of these vesicles
could be amenable to therapeutic interventions.14,51
Sources of Funding
This work was supported by National Institutes of Health (USA)
grants HL56984 and HL73898 (to K.J.W.) and HL69471 and
HL081503 (to M.P.R.).
Disclosures
None.
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Cholesterol Enrichment of Human Monocyte/Macrophages Induces Surface Exposure of
Phosphatidylserine and the Release of Biologically-Active Tissue Factor−Positive
Microvesicles
Ming-Lin Liu, Michael P. Reilly, Peter Casasanto, Steven E. McKenzie and Kevin Jon Williams
Arterioscler Thromb Vasc Biol. 2007;27:430-435; originally published online December 7,
2006;
doi: 10.1161/01.ATV.0000254674.47693.e8
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SUPPLEMENTAL FIGURES
for
Cholesterol enrichment of human monocyte/macrophages induces surface exposure of
phosphatidylserine and the release of biologically active, tissue factor-positive
microvesicles
Ming-Lin Liu*, Michael P. Reilly†, Peter Casasanto†, Steven E. McKenzie†,
and Kevin Jon Williams*
From the *Dorrance H. Hamilton Research Laboratories, Division of Endocrinology, Diabetes
and Metabolic Diseases, and the †Cardeza Foundation for Hematologic Research, Division of
Hematology, Department of Medicine, Jefferson Medical College of Thomas Jefferson
University, Philadelphia, PA 19107, U.S.A.
1
Supplemental Figure I
Supplemental Figure I. Cholesterol enrichment of monocytic cells increases TF antigen release.
Suspensions of human THP-1 monocytes were treated for 4h at 37ºC with UC, LPS, both, or
neither (Control), as indicated. Displayed are TF ELISA data from culture supernatants prepared
from the same samples as in Figure 2A. P<0.01 by ANOVA. †P<0.01 and ‡P<0.001 vs the control
group by the Student-Newman-Keuls test.
2
Supplemental Figure II
Supplemental Figure II. Cholesterol enrichment of primary human monocyte-derived
macrophages increases the generation of TF-positive microvesicles. Adherent primary human
MDMs in 6-well plates were treated without (Control) or with UC for 20h. Displayed are
quantitative counts of PS-positive MVs that were also TF-positive, from the same samples as in
Figure 3. ‡P<0.001 vs the control group by Student’s unpaired t-test.
3
Supplemental Figure III
Supplemental Figure III. Cholesterol enrichment of monocytes increases cell-surface display
of PS. THP-1 monocytes were incubated at 37ºC with or without UC, LPS, or combinations, as
indicated, then analyzed by flow cytometry. Displayed are representative histograms of annexinV staining, gated to include only cells, after 0h (red), 2h (black), 4h (green), and 20h (blue),
from the same samples as in Figure 4. Each histogram includes approximately 1000 cells.
4
Supplemental Figure IV
5
Supplemental Figure IV. Role for apoptosis in cholesterol-induced microvesicle generation.
THP-1 monocytic cells were incubated at 37ºC without or with UC, Z-DEVD-FMK (a caspase-3
inhibitor), or combinations, as indicated, then analyzed by flow cytometry. Panel A displays
representative dot-plots for UC-treated THP-1 cells without or with Z-DEVD-FMK. Populations
of MVs and cells are indicated as in Figure 1A. Panel B shows quantitative counts of TUNELpositive cells after the indicated treatments, to demonstrate a significant inhibition of UCinduced apoptosis by the caspase-3 inhibitor. In panel B, P<0.01 by ANOVA. Symbols labeled
with different lowercase letters are statistically different by the Student-Newman-Keuls test
(P<0.05). Results are displayed from the same samples as in Figure 5.
6